Delivering a toxic metal to the active site of urease

Urease is a nickel (Ni) enzyme that is essential for the colonization of Helicobacter pylori in the human stomach. To solve the problem of delivering the toxic Ni ion to the active site without diffusing into the cytoplasm, cells have evolved metal carrier proteins, or metallochaperones, to deliver the toxic ions to specific protein complexes. Ni delivery requires urease to form an activation complex with the urease accessory proteins UreFD and UreG. Here, we determined the cryo–electron microscopy structures of H. pylori UreFD/urease and Klebsiella pneumoniae UreD/urease complexes at 2.3- and 2.7-angstrom resolutions, respectively. Combining structural, mutagenesis, and biochemical studies, we show that the formation of the activation complex opens a 100-angstrom-long tunnel, where the Ni ion is delivered through UreFD to the active site of urease.


INTRODUCTION
Urease is a virulence factor of Helicobacter pylori that infects half of the human population, leading to an increased risk of peptic ulcers and gastric cancer (1). The enzyme, hydrolyzing urea into carbon dioxide and ammonia, is essential to the survival of the pathogen in the acidic environment of the human stomach (2). Most ureases are nickel (Ni) enzymes containing two Ni(II) ions bridged by a carbamylated lysine residue (Lys 219 of H. pylori urease) in their active sites (3)(4)(5)(6). Metal ions at the top of the Irving-Williams series (7) such as Ni(II) ions are toxic because they can displace weaker ions [e.g., Mg 2+ in guanosine triphosphatase (GTPase)] from the active site of cellular enzymes and can inactivate these enzymes (8).
The urease maturation pathway represents a paradigm for how cells solve the problem of delivering a toxic metal ion to the active site of an essential enzyme. Cells have evolved metallochaperones, or metal carrier proteins, to deliver the Ni(II) ions from one protein to another via the formation of specific protein-protein complexes so that the toxic metal ions do not diffuse into the cytoplasm (9). In the urease maturation pathway, Ni delivery is assisted by four urease accessory proteins UreD, UreE, UreF, and UreG (fig. S1) (9)(10)(11). UreE exists as a homodimer in solution containing the conserved Gly-Asn-Arg-His motif that binds a Ni(II) ion at the dimeric interface (12)(13)(14). UreG is a GTPase that undergoes guanosine triphosphate (GTP)-dependent dimerization that brings the conserved Cys-Pro-His (CPH) motif to the dimer interface to bind a Ni(II) ion in a square planar coordination (15). UreD interacts with UreF to form a dimer of heterodimeric UreFD complex, which induces conformational changes in UreF to recruit a UreG dimer to form a UreGFD complex (16,17). UreE provides the upstream source of Ni(II) ions, which are delivered to UreG via the formation of a UreE 2 G 2 heterodimeric complex (15,18,19). After receiving its Ni, the Ni-bound UreG dimer forms an activation complex with UreFD and urease, and activates urease upon GTP hydrolysis with the help of UreFD (Fig. 1A) (15,17,20).
How Ni is transferred from UreG to urease remains elusive. The Ni binding site of urease is deeply buried (3,4), so it is not known how Ni can access the urease active site. Moreover, because UreG and the urease are topologically separated by the UreFD complex, the Ni binding site of UreG will likely be far away from the urease (Fig. 1A). It is also not known what could be the role of the UreFD complex in this process. In this study, we determined the cryo-electron microscopy (cryo-EM) structures of H. pylori HpUreFD/urease complex and Klebsiella pneumoniae KpUreD/urease complex. We show that formation of the UreFD/urease complex opens a tunnel from the buried urease active site that can connect to the Ni binding site in UreG. Further supported by mutagenesis and biochemical studies, we suggest that this tunnel facilitates the delivery of Ni(II) ion from UreG to the urease.
The EM maps of HpUreFD/urease and KpUreD/urease were solved to 2.3-and 2.7-Å resolution, respectively ( Fig. 1 and fig.  S2). Cartoon representation and secondary structure assignment of the modeled structures are shown in the figs. S3 to S5. In K. pneumoniae urease, each catalytic unit is constituted by the α (KpUreC), β (KpUreB), and γ (KpUreA) domains (Fig. 1D). Similar to the quaternary structure of Klebsiella aerogenes urease (3), K. pneumoniae urease contains three catalytic units arranged in a C3 trimer that resembles a triangular disc-like structure (Fig. 1B). The vertexes of the urease trimer are bound with a KpUreD molecule (Fig. 1, B and D) that has the characteristic β helix fold ( fig. S3). In H. pylori urease, each catalytic unit is constituted by HpUreA (a fusion of the β and γ domains) and HpUreB (α domain) ( Fig. 1E and figs. S4 and S5). Four copies of the urease trimers form a dodecameric quaternary structure with 12 protruding HpUreFD molecules (Fig. 1C). As expected, the R179A/Y183D substitutions disrupted the dimerization of HpUreFD, and each catalytic unit of H. pylori urease is interacting with one copy of HpUreD and HpUreF (Fig. 1E). As for the KpUreD/urease complex, HpUreD interacts with the urease (Fig. 1E). The binding sites of KpUreD and HpUreFD on urease are very similar and involve an area of the α domain flanked by the β and γ domains (Fig. 1, F and G). The surface area buried by UreD on K. pneumoniae and H. pylori ureases were 2225 and 2384 Å 2 , respectively. Residues involved in polar contacts (hydrogen bonds or salt bridges) between UreD and the urease are indicated in Fig. 1 (F and G) and listed in table S1.

Complex formation induces conformational changes in urease and UreD
The structure of the HpUreFD/urease complex was compared to the crystal structures of HpUreFD (16) and H. pylori native urease (4). In addition to minor structural changes at the C terminus of helix 1 in the dimerization interface of HpUreF, residues with large values of Cα RMSD (root mean square deviation of α carbon) are found in the binding interface between HpUreD and HpUreB ( Fig. 2A). The conformational changes upon the formation of HpUreD/urease complex are summarized in Fig. 2 and in movie S1. Binding of UreD induces major conformational changes in three switch regions of HpUreB: (i) the glycine-rich loop ( 277 GAGGGHAP 284 ) between strand 6 and helix 6, (ii) helix H3 and the loop connecting to helix 7 (residues 330 to 340), and (iii) residues between strand 12 and strand 13 (residues 538 to 545) (Fig. 2B). Upon binding of HpUreFD, residues 335 ADSR 338 at the C terminus of helix H3 uncoil, causing switches II and III residues to stretch in opposite directions and residues Arg 338 and Arg 340 to flip out toward UreD (Fig. 2B). As Arg 338 is hydrogen bonded to the backbone amide of Ala 278 and Gly 279 , the conformational change is propagated to the glycine-rich switch I loop (Fig. 2B). When the structure of the KpUreD/urease complex was compared to the crystal structure of K. aerogenes urease apoprotein (3) and native urease (21), similar conformational changes were observed ( Fig. 2C and fig. S6).
The formation of the HpUreFD/urease complex also induces major conformational changes in HpUreD ( Fig. 2F and movie   (Fig. 2F). On the other hand, Gln 80 undergoes a swivel motion (Fig. 2F) so that it can form hydrogen bonds to the backbone amides of Val 332 and Ala 335 of HpUreB (Fig. 2G). This swivel motion induces structural rearrangement in the regions of strand 6/7 and strand 9/10 in such a way that the side chain of Phe 112 swings out from a buried to an exposed position (Fig. 2F). The backbone conformations of the loops are stabilized by additional hydrogen bonds involving Ile 77 , Ser 79 , Ser 81 , Ala 110 , and Phe 112 as indicated in Fig. 2G.

UreD/urease interaction is important in urease maturation
The most notable conformational changes observed in urease are the flipping of Arg 338 and Arg 340 toward UreD. The conformation of Arg 338 is stabilized by forming a hydrogen bond to and stacking against Tyr 543 of HpUreB (Fig. 3A). On the other hand, Arg 340 of HpUreB forms an intermolecular hydrogen bond with Asp 61 of HpUreD (Fig. 3A). The interaction between HpUreD and urease is further strengthened by Glu 177 of HpUreA forming hydrogen bonds with Phe 82 and Lys 84 of HpUreD. These hydrogen bonds are also conserved in KpUreD/urease ( fig. S7). To test the role of these conserved interactions in urease maturation, we have created a D61A variant of HpUreD, E177A variant of HpUreA, and Y543A variant of HpUreB and tested the interaction between HpUreFD and HpUreAB via a pull-down assay (Fig. 3, B and C).
Our results show that wild-type (WT) polyhistidine glutathione Stransferase (HisGST) tagged HpUreFD coelutes with HpUreAB, suggesting that HpUreFD interacts with HpUreAB in the pulldown assay ( Fig. 3B and fig. S8). In contrast, the interaction between HpUreFD and HpUreAB was greatly reduced by D61A substitution of HpUreD (Fig. 3B), E117A substitution of HpUreA, and Y543A substitution of HpUreB (Fig. 3C). We further show that these substitutions abolish urease activity in an in vitro assay (Fig. 3, D and E). These results suggest that these conserved polar interactions are important in the formation of the HpUreFD/urease complex and in the maturation of urease.

A tunnel inside the HpUreFD/urease complex facilitates urease maturation
We noticed that the formation of the HpUreFD/urease complex opens a tunnel that reaches the active site of urease (Fig. 4A). Tunnel searching was performed using the program CAVER 3.0 (22). This 100-Å-long tunnel starts at the active site residue Lys 219 of urease, exits HpUreB near Asp 336 of the switch II region, passes through HpUreD between the two layers of β sheets, enters HpUreF near Ala 233 , and reaches the dimerization interface of HpUreF (Fig. 4A). A tunnel was also identified in the KpUreD/urease complex that passes through similar regions in KpUreC and KpUreD ( fig. S9). In the crystal structure of the HpUreGFD complex (17), a tunnel that passes through a similar region of HpUreF was identified ( Fig. 4B) (23)(24)(25). Together, these observations suggest that the tunnel inside the HpUreFD/urease complex connects the active site of urease ( Fig. 4A) to the CPH Ni binding motif of UreG (Fig. 4B).
Conformational changes in the HpUreB/UreD interface are instrumental in the opening of the tunnel that reaches the urease active site ( Fig. 4C and movie S2). The active site residue Lys 219 , which is carbamylated and binds Ni(II) ions in mature urease, is completely buried inside urease. The access to the active site is blocked by the glycine-rich switch I loop and switch II residues (e.g., Phe 334 and Arg 338 ) of HpUreB (Fig. 4C). These residues relocate upon the formation of the HpUreFD/urease complex, making room to create a tunnel that reaches the active site of urease. On the other hand, the tunnel in the HpUreD is blocked by Phe 112 before the formation of the HpUreFD/urease complex (Fig. 4C). As described above (Fig. 2F), the UreD/urease interaction causes Phe 112 to flip to an exposed position and thereby open a passage that connects the tunnel between HpUreB and HpUreD (Fig. 4C).
To test whether the tunnel is essential for Ni delivery to the urease, we introduced tunnel-disrupting substitutions to residues along the tunnel. We first introduced charge-to-alanine substitutions in acidic residues buried inside the tunnel (i.e., D336A of HpUreB, E140A of HpUreD, and E85A of HpUreF). We hypothesized that these conserved acidic residues (refer to the alignment in figs. S3 and S5) are important in stabilizing the positively charged Ni(II) ions inside the tunnel. The second strategy was to introduce lysine substitutions to small amino acid residues along the tunnel (i.e., S81K of HpUreD and A41K, S47K, and A233K of HpUreF) because our modeling suggested that the flexible lysine side chain can block the tunnel without affecting the proper folding of HpUreFD.
We first coexpressed HpUreD str with HpUreF and HpUreG in E. coli and purified the resulting HpUreGFD str complex by Strep-Tactin affinity chromatography (fig. S10A). We show that the tunnel-disrupting substitutions in HpUreD (S81K and E140A) and HpUreF (A41K, S47K, E85A, and A233K) did not affect the formation of the HpUreGFD str complex (fig. S10, C and E), and the purified HpUreGFD str interacted with WT HpUreAB in a pulldown assay (Fig. 4D). The interaction was not affected by tunneldisrupting substitutions in HpUreB (D336A; Fig. 4D), HpUreD (S81K and E140A; Fig. 4E), and HpUreF (A41K, S47K, E85A, and A233K; Fig. 4F). These observations suggest that the tunnel-disrupting substitutions do not disrupt formation of the activation complex between the urease and its accessory proteins. On the other hand, all these tunnel-disrupting substitutions abolished urease activity (Fig. 4, G to I). As shown in Fig. 4 (A and B), these tunnel-disrupting substitutions are distributed along the 100-Å-long tunnel-D336A and S81K are located at the HpUreB/ UreD interface, E140A is located inside HpUreD, A233K is located at the HpUreD/UreF interface, E85A is located inside HpUreF, and A41K and S47K are located at the dimeric interface of HpUreF. Together, our results suggest that Ni(II) ions are delivered along this tunnel, passing through HpUreF and HpUreD, to reach the active site of urease. HpUreAB was activated with (WT/variant) HpUreFD and HpUreG in the reaction buffer at 37°C for 30 min. Ni 2+ was omitted in the reaction buffer in the negative control. Mean relative activity and SEM of at least three measurements were reported. Urease activities of D61A, E177A, and Y543A variants of HpUreD, HpUreA, and HpUreB, respectively, were significantly lower than that of WT [one-way analysis of variance (ANOVA), P < 0.0001]. There were no significant differences among variants and the negative control. To test whether urease maturation requires dimerization of UreFD, we performed the urease activation assay in two-chamber dialyzers using the strategy described previously ( fig. S11) (15). In this assay, Ni-bound HpUreG provides the sole source of Ni for urease activation. Our results showed that urease was activated when Ni-bound HpUreG was mixed with WT HpUreFD and HpUreAB in the same chamber ( fig. S11A). On the other hand, when Ni-bound HpUreG was separated from HpUreFD and HpUreAB by a dialysis membrane, urease activation was abolished ( fig. S11D). Substitutions of R179A/Y183D in HpUreF, which breaks dimerization of HpUreFD, abolished urease activation in vitro ( fig. S11B). We have previously showed that the R179A/ Y183D substitutions abolished the formation of HpUreGFD complex and inhibited urease maturation in vivo (17). Together, our results suggest that Ni delivery requires HpUreG to interact with the dimeric HpUreFD in complex with urease.

DISCUSSION
To avoid cytotoxicity, ions such as copper and Ni are tightly regulated to subnanomolar concentrations (corresponding to less than one ion per bacterial cell) (26). To activate the urease, the enzyme cannot just pick up free Ni(II) ions from the cytoplasm. Instead, the Ni(II) ions are acquired by metallochaperones such as UreE and UreG and are delivered to the active site of urease within specific protein complexes so that the toxic metal ions do not diffuse into the cytoplasm. After receiving its Ni(II) ions from the UreE 2 G 2 complex (15,18), Ni-bound UreG activates urease by forming a UreGFD/urease activation complex (17,20). When Ni-bound UreG was separated from the UreFD/urease complex by a dialysis membrane in a two-chamber dialyzer, the urease activation was abolished (15). This observation suggests that direct proteinprotein interaction between UreG and UreFD/urease is essential for Ni delivery from UreG to the urease (15). The dimerization-deficient variant of UreF(R179A/Y183D) failed to form the UreGFD complex and to activate urease in vitro (fig. S11) and in vivo (17), HpUreAB eluted in all variants tested, but not in the vector control. (G to I) In vitro urease activation assay. HpUreAB (WT/variant) was activated with (WT/variant) HpUreGFD str in the reaction buffer at 37°C for 30 min. Ni 2+ was omitted in the reaction buffer in the negative control. Mean relative activity and SEM of at least three measurements were reported. Urease activities of all tunnel-disrupting variants were significantly lowered than that of WT (one-way ANOVA, P < 0.0001). There were no significant differences among variants and the negative control. (J) A model of how Ni is delivered through a tunnel from UreG to the urease. Phe 112 of HpUreD serves as a gate residue that relocates from a buried to an exposed position, opening a passage to the urease active site. Upon GTP hydrolysis, the Ni(II) ion is released from UreG, enters the protein tunnel that passes through UreF and UreD, and reaches the urease active site. suggesting that UreG delivers its Ni through interaction with the UreFD dimer and the urease. Ni-bound UreG is likely interacting with UreF in the activation complex, as substitutions that break UreG/UreF interaction also abolish urease maturation (16,27).
We have modeled the structure of the HpUreGFD/urease activation complex based on the cryo-EM structure of HpUreFD/urease (this study) and the crystal structure of the guanosine diphosphate (GDP)-bound HpUreGFD complex ( fig. S12) (17) to propose a model of how Ni is delivered from UreG to urease (Fig. 4J). A tunnel can be identified in the activation complex that connects the active site of urease to the Ni binding site of UreG ( fig. S12). In the GTP-bound state of UreG, the Ni(II) ion is bound at the dimer interface by Cys 66 and His 68 of the UreG-conserved CPH motif (15). UreG is modeled in the GDP-bound state and may represent the structure of the activation complex immediately after GTP hydrolysis, which disrupts the Ni-binding square-planar coordination ligands ( fig. S12). Our mutagenesis and biochemical studies suggest that Ni released from UreG can pass through UreF to UreD to reach the urease buried active site via this tunnel (Fig. 4J).
A protein tunnel can also be defined with the HpUreGFD complex and predicted in K. aerogenes UreD (17,(23)(24)(25)28). The tunnel in HpUreGFD follows a similar path to that identified in the HpUreFD/urease complex until it reaches the region near Glu 140 of HpUreD. Molecular dynamics simulations suggest that hydrated Ni(II) ions can pass through this tunnel from the Nibinding CPH motif of UreG to Glu 140 of UreD inside the HpUr-eGFD complex (25). Comparing the structures of the HpUreFD/ urease and HpUreGFD complexes reveal that Phe 112 of HpUreD can serve as a gate residue that blocks the tunnel in the HpUreGFD complex (Fig. 4C).
The HpUreD gate residue only opens to allow access to the urease active site following the conformational changes in HpUreD and HpUreB induced by formation of the activation complex ( Fig. 4C and movie S2). In particular, the gate residue Phe 112 relocates from a buried position to an exposed position, thus "opening" the tunnel to allow passage of Ni(II) ions to the urease active site (Fig. 4J). On the other hand, Phe 112 of HpUreD on the other side of the activation complex, away from the urease, is expected to adopt the buried position that "closes" the tunnel (Fig. 4J). The closure of the tunnel ensures the Ni(II) ion does not diffuse into the cytoplasm at the other end of the complex. Our model also explains how GTP hydrolysis thermodynamically drives the Ni delivery to the ureasebecause GTP hydrolysis disrupts the Ni binding site at UreG, the released Ni(II) ion is trapped inside the activation complex until it reaches the active site of urease, where it can form more stable interactions.
The activation complex should dissociate after urease is activated. In native H. pylori urease, the Ni(II) ions are coordinated by carbamylated Lys 219 , His 136 , His 138 , His 274 , and Asp 320 (fig. S13A). Structural comparison reveals that Ni binding to the active site relocates His 274 in a position to form a hydrogen bond to Gly 280 of switch II (fig. S13A). This interaction promotes conformational changes in switches I and II such that Arg 338 flips back toward the active site to form hydrogen bonds to Ala 278 and Gly 279 , inducing dissociation of UreD from the urease. Similar conformational changes are also conserved in Klebsiella urease (fig. S13B). These observations suggest that Ni binding to the active site provides additional interactions that promote dissociation of the activation complex.
In summary, this study shows how cells solve the problem of delivering a toxic metal ion to an essential enzyme by opening a 100-Å-long tunnel within the activation complex so that the toxic Ni(II) ion is delivered through the tunnel to the urease active site. The urease maturation pathway thus provides a paradigm on trafficking of toxic metal ions in cells. The delivery of Ni(II) ions along the urease maturation pathway always occurs within protein complexes so that the toxic metal ions cannot escape into the cytoplasm. In addition to academic curiosity, because H. pylori requires active urease to survive in the acidic environment of human stomach (2), a better understanding of the mechanism of the urease maturation pathway could provide insights into the development of treatment for H. pylori infection (29). Figure S14 summarizes the constructs used in this study. The construction of pHpA2H and introduction of R179A/Y178D substitutions to HpUreF were described previously (14,15). To create pHpA2H str -UreF(R179A/Y183D)UreD(E140A), E140A substitution and a C-terminal Strep-tag II (WSHPQFEK) were added to HpUreD (encoded by ureH). To create the plasmid pKpUreD str ABC, the K. pneumoniae gene cluster ureDABC was cloned between the Nde I and Xho I sites of the pRSF-Duet1 vector (Novagen) with a Strep-tag II fused to the N terminus of KpUreD. The construction of plasmids pHisSUMO-HpUreG, pHisGST-HpUreF, pHpUreH, and pHpUreAB (encoding HisSUMO-HpUreG, HisGST-HpUreF, HpUreD, and HpUreAB, respectively) used in this work was described previously (16,17). To create the plasmid pHpUreGFD str , the H. pylori gene cluster ureFGH was cloned between the Nde I and Eco RI sites of an inhouse pRSETA (Invitrogen) vector with a Strep-tag II fused to the C terminus of HpUreD. To create the plasmid pHisGST, the coding sequence of the GST was cloned between the Eco R1 and Pas I sites of the pET-Duet1 vector (Novagen) with an N-terminal polyhistidine tag. Variants of HpUreA, HpUreD, and HpUreF were generated using the Q5 mutagenesis kit (New England Biolabs) or the QuikChange II site-directed mutagenesis kit (Agilent) following the protocols from the manufacturers. Variants of HpUreB were generated by overlap extension polymerase chain reaction (30). Primers used for site-directed mutagenesis are listed in the table S2.

Structure determination of the HpUreFD/urease and
KpUreD/urease complexes Protein sample preparation E. coli BL21 was transformed with the expression plasmids of pHpA2H str -UreF(R179A/Y183D)UreD(E140A) and pKpUreD str ABC, cultured in LB at 37°C, and induced overnight with 0.4 mM isopropyl β-D-thiogalactopyranoside (IPTG) when optical density at 600 nm (OD 600 ) reached 0.5. The harvested cells were resuspended in the Strep-binding buffer [50 mM Hepes, 200 mM NaCl, and 0.5 mM TCEP (pH 7.5)] and lysed using EMULSIFLEX-C5. The soluble lysate was loaded onto a Strep-Tactin XT column (IBA Lifesciences), washed extensively with the Strep-binding buffer, and eluted with 50 mM D-biotin in the Strep-binding buffer. The HpUreFD/urease and KpUreD/urease complex were further purified by size exclusion chromatography using a Superose-6 10/300 column (GE Health-Care) pre-equilibrated with the Strep-binding buffer. Quantifoil R1.2/1.3 copper grids (200 mesh) with holey carbon foil were glow discharged for 20 s at 15 mA. Four microliters of KpUreD/ urease (0.2 mg/ml) or HpUreFD/urease complex (0.5 mg/ml) was applied to the grids. Grids were plunge-frozen in liquid ethane using the Vitrobot Mark IV (Thermo Fisher Scientific) maintained at 100% humidity at 4°C, with blot time of 3 s and blot force of 0.

Collection and processing of cryo-EM data
For the structure determination of the HpUreFD/urease complex, 1953 movies were collected using a K2 detector on a Titan G3 microscope at a calibrated pixel size of 0.822 Å. Each movie was imaged with a total dose of 48e − /Å 2 . Preprocessing was streamed using SIMPLE3.0 (31) including patched motion correction (5 × 5 patches), patched contrast transfer function (CTF) correction (5 × 5 patches), and auto picking using a template derived from twodimensional (2D) averages generated from 200 handpicked particles from a preliminary run. A total of 166,893 particles were picked. Two rounds of 2D classification led to a set of 132,431 particles, and an initial model was generated from the 2D class averages using SIMPLE3.1 with tetrahedral symmetry imposed. The particles were exported to RELION3.0 (32), and 3D classification into three classes led to identification of a subset of 68,516 particles with higher occupancy for the UreFD components. Further rounds of 3D refinement, CTF refinement, and particle polishing led to a volume of 2.3-Å resolution [as assessed by gold standard Fourier shell correlation (FSC) = 0.143 criterion].
For the structure determination of the KpUreD/urease complex, 3149 movies were collected using a K2 detector on a Titan G3 microscope at a calibrated pixel size of 0.822 Å. Each movie was imaged with a total dose of 48e − /Å 2 . Preprocessing was streamed using SIMPLE3.0 including patched motion correction (5 × 5 patches), patched CTF correction (5 × 5 patches), and auto picking using a template derived from 2D averages generated from~200 handpicked particles. A total of 567,251 particles were picked. Rounds of 2D classification led to a set of 476,622 particles, and an initial model was generated in SIMPLE3.0 from the 2D class averages. 3D classification within RELION3.0 into six classes led to identification of a subset of 111,242 particles with higher occupancy of the UreD component on all three vertices. Additional rounds of 2D classification pruned this subset to 89,857 particles before further rounds of 3D refinement with C3 symmetry imposed, CTF refinement, and particle polishing, leading to a volume of 2.7-Å resolution (as assessed by gold standard FSC = 0.143 criterion).

Model building and refinement
Initial models for the KpUreD/urease complex were derived from the crystal structure of K. aerogenes urease apoprotein [Protein Data Bank (PDB): 1KRA] (3), and a KpUreD model was generated by homology modeling using the program MODELLER implemented in UCSF CHIMERA (33). Initial models for the HpUreFD/urease complex were derived from the crystal structure of H. pylori native urease (PDB: 1E9Z) (4) and the structure of HpUreFD from the HpUreGFD complex (PDB: 4HI0) (17). These initial models were fitted into the cryo-EM maps of the KpUreD/ urease and the HpUreFD/urease complexes using the program UCSF CHIMERA (34). Models were built interactively using the program COOT (35) and refined using the program PHENIX.REAL_SPACE_REFINE (36).

Structure analysis
Tunnel searching was performed by the program CAVER 3.0 (22) implemented in the program PyMOL (https://pymol.org) using the default setting of a 0.9-Å probe radius. The structures of the HpUreFD/urease and KpUreD/urease complexes were superimposed to the native structure of H. pylori and K. aerogenes ureases (PDB: 1E9Z and 1FWJ) to identify the location of the Ni binding sites, which serve as the starting point of the tunnel search. The movies S1 and S2 were created using the program PyMOL to morph the structures of HpUreAB (PDB: 1E9Z) and HpUreFD (PDB: 3SF5) into the structure of HpUreFD/UreAB (this study). To prepare cell lysates expressing WT and variants of HpUreAB, E. coli Rosetta (DE3) was transformed with pHpUreAB (WT/variants), cultured in King Broth or Terrific Broth with appropriate antibiotics [kanamycin (50 μg/ml) and chloramphenicol (25 μg/ml), induced overnight with 0.4 mM IPTG when OD 600 reached 0.6 to 0.8 at 25°C. Each gram of cell pellet was resuspended in 10 ml of the assay buffer and lysed by sonication. After centrifuging at 20,000g for 30 min, the soluble lysates were collected and filtered using 0.22μm filters. The concentrations of WT and variants of HpUreAB in the lysates were normalized according to Coomassie Blue staining ( fig. S8D).

Pull-down assay
Five milliliters of protein samples of (WT/variant) HisGST-HpUreFD or HisGST at 20 μM was loaded onto 5-ml GSTrap FF columns (GE HealthCare) and incubated at 37°C for 30 min. After the columns were washed with 30 ml of assay buffer, 5 ml of lysates of E. coli expressing (WT/variant) HpUreAB was added to the columns and incubated at 37°C for another 30 min. The columns were washed with 30 ml of assay buffer, eluted with 10 mM GSH in the assay buffer, analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Blue (Fig. 3, B and C).

Pull-down assay for testing interactions between HpUreGFD str and H. pylori urease Protein sample preparation
To purify protein samples of WT or variants of HpUreGFD str , E. coli BL21 (DE3) pLysS was transformed with pHpUreGFD str (WT/variants), cultured in Terrific Broth with appropriate antibiotics [ampicillin (100 μg/ml) and chloramphenicol (25 μg/ml), induced overnight with 0.4 mM IPTG when OD 600 reached 0.6 to 0.8 at 25°C. One gram of harvested cells was resuspended in 10 ml of Strep-binding buffer [50 mM Hepes, 200 mM NaCl, and 1 mM TCEP-HCl (pH 7.5)] supplemented with 0.1 g of cOmplete ULTRA Tablets protease inhibitor cocktail (Roche). After lysis by sonication, the cell lysate was collected by centrifugation at 20,000g for 40 min and filtered using 0.22-μm filters. A total of 0.5 mM GDP and 1 mM MgSO 4 were added to the cell lysate, which was then loaded onto 0.5-ml Strep- Tactin

Pull-down assay
A total of 0.1 ml of 20 μM HpUreGFD str (WT/variants) was loaded onto 0.1-ml Strep-Tactin XT resins (IBA Lifesciences) pre-equilibrated with the Strep-binding buffer in a spin column. After washing the resins with 8 bed volumes of the assay buffer, 100 μl of lysate of HpUreAB (WT/variants) was loaded onto the resins. After incubation at 37°C for 15 min, the resins were washed with 13 bed volumes of the assay buffer. The last trace of buffer was removed by centrifugation at 100g for 10 s. Bound proteins were eluted by 0.25 ml of the Strep-elution buffer, analyzed by SDS-PAGE, and stained with Coomassie Blue (Fig. 4, D to F).
After sonication, cell lysate was loaded onto a HiTrap Q HP column (GE HealthCare) pre-equilibrated with buffer A. The column was washed with buffer A and eluted with a 100-ml linear gradient of 0 to 500 mM NaCl in buffer A. Fractions corresponded to~200 to 325 mM NaCl were collected and concentrated to OD 280 of~24 to 27. Two hundred fifty microliters of the sample was loaded onto a Superdex 200 Increase 10/300 column (GE HeathCare) pre-equilibrated with the assay buffer [20 mM Hepes, 200 mM NaCl, and 1 mM TCEP-HCl (pH 7.5)]. Fractions that correspond to the HpUreAB dodecamer (~10.5 ml) were collected, concentrated, and loaded onto a Superose 6 Increase 10/300 column (GE HeathCare) pre-equilibrated with the assay buffer. Purified HpUreAB dodecamer was eluted at~13.5 ml.

Urease activation assay
For the urease activation assay in Fig. 3 Fig. 4, 40 μM HpUreGFD str (WT/variants) were mixed with 10 μM HpUreAB (WT/variants) in the reaction buffer instead. NiSO 4 was omitted in the negative control. KHCO 3 (10 mM) was added to stimulate the GTP hydrolysis of HpUreG (17,18) and initiate the urease activation. The reaction mixture was incubated at 37°C for 30 min. Activation of urease was measured as described (15). For the urease activation assay in fig. S11, 40 μM Ni-bound or apo-HpUreG, (WT/variant) HpUreFD, and 10 μM HpUreAB were added to either side of a two-chamber dialyzer (Bioprobes Ltd.) separated by a dialysis membrane with a molecular weight cutoff of 6 to 8 kDa (Spectrum Labs). The buffer in both chambers contained 2 mM MgSO 4 , 1 mM GTP, 20 mM Hepes (pH 7.5), 200 mM NaCl, and 1 mM TCEP. After equilibration at 4°C for 16 hours, 10 mM KHCO 3 was added to both chambers to activate the GTP hydrolysis required for urease activation. The chambers were then incubated at 37°C for 1 hour, and the urease activity was measured as described (15). Urease activities were normalized to those measured for using WT proteins and were analyzed with one-way analysis of variance (ANOVA) followed by the Tukey post hoc test using the program Prism (Graph-Pad). In our hand, specific activity of activated urease ranged from 27 to 181 μmol min −1 mg −1 (table S3). In comparison, the specific activity of native urease purified from H. pylori was 1693 μmol min −1 mg −1 (37).

Supplementary Materials
This PDF file includes: Figs. S1 to S14 Tables S1 to S3 Legends for movies S1 and S2 Other Supplementary Material for this manuscript includes the following: Movies S1 and S2